acrylamide polymerization

tfitzwater at nexstar.com tfitzwater at nexstar.com
Mon Sep 22 11:33:17 EST 1997


(subscript: The best source for information about acrylamide 
polymerization
is   BioRad   Bulletin   1156:    "Polyacrylamide   gels   are   formed  
by
co-polymerization  of  acrylamide  and  bis-acrylamide.   The reaction is 
a
vinyl  addition  polymerization  initiated  by  a  free  
radical-generating
system.   Polymerization is initiated by TEMED 
(tetramethylethylenediamine)
and  APS  (ammonium  persulfate).  The TEMED acts as an electron carrier 
to
activate  the acrylamide monomer, providing an unpaired electron to 
convert
the  acrylamide  monomer  to  a  free  radical.  The activated monomer 
then
reacts  with  unactivated monomer to begin the polymer chain reaction.  
The
elongating  polymer  chains  are  randomly crosslinked by bis, resulting 
in
closed loops and a complex web polymer with a characteristic porosity 
which
depends on the polymerization conditions and monomer concentration." )
(subscript:  Gels  may not completely polymerize, depending on factors 
such
as  concentration  of  initiator,  temperature,  pH,  additives,  
breakdown
products  and  impurities  in the chemicals and water used.  Light and 
high
temperature can cause autopolymerization of linear acrylamide which 
affects
the reproducibility of gel formation.)
(subscript:  )
(subscript: Acrylamide purity)
(subscript:  Poor  quality  acrylamide contains acrylic acid (a 
deamidation
product)  which  will  copolymerize  and  cause local pH changes in the 
gel
leading   to  streaking  and  smearing  of  bands.   Linear  
polyacrylamide
contaminants  effect  reproducibility.  Ionic contaminants can include 
both
inhibitors and accelerators of polymerization.)
(subscript:  During  storage  at  room  temperature,  especially  in 
water,
acrylamide breaks down into acrylic acid, polyacrylamide, )(subscript: 
BBB)
(subscript: -nitrolotrispropionamide and ammonia.  The pH of 10% 
acrylamide
dissolved   in   0.1  N  NaCl  should  be  6.50  +/-  0.5   The  pH  of  
1%
N,N'-Methylene-Bis-Acrylamide  dissolved  in  Type  I  water  should  be  
)
(subscript: >)(subscript:  5.5.)

(subscript: Urea)
(subscript:  Urea (NH2)2CO has a formula weight of 60.06.  The problem 
with
the  use  of  urea  in  protein gels is the accumulation of cyanate ions 
in
stock  solutions  due  to  chemical isomerization.  The cyanate reacts 
with
amino  groups  to  form  stable  carbamylated  derivatives which modify 
the
charge  of  proteins.  The free amino groups of Tris neutralize the 
cyanate
ions.   Where  possible, the heating of urea-containing solutions should 
be
avoided   as   cyanate   ions  formation  is  accelerated  with  
increasing
temperature.   Urea  slowly  decomposes  in water, forming ammonia and 
very
reactive  cyanate  ions.   Urea  solutions may be pretreated with 
mixed-bed
resins  or  mixed  with  small amounts of Tris that reacts with the 
cyanate
ions.  )

(subscript: Tris)
(subscript:  It is essential that Tris buffers are prepared with Tris 
base.
After  Tris base has been dissolved in Type I water, the pH of the 
solution
should  be  adjusted  with  HCl.  If Tris-HCl or Trizma are used to 
prepare
buffers,  the  concentration  of  salt  will  be too high and material 
will
migrate anomalously, yielding extremely diffuse bands.  )
(subscript:  Tris-base is oxidized during electrophoresis to a 
UV-absorbing
compound.)
(subscript:   )
(subscript: APS (ammonium persulfate))(subscript:  MW 228.22)
(subscript:  APS  is  a strong oxidant.  It is an irritant by inhalation 
or
contact with skin or eyes.  )

(subscript:  APS  (ammonium persulfate) is very hygroscopic and reactive 
by
nature  and  is  prone  to oxidation and decomposition.  It begins to 
break
down  almost immediately when dissolved in water.  Optimally, APS should 
be
prepared fresh daily.  Certainly, 10% stocks should be replaced every 
month
for  minimal  reproducibility.   Listen  for  a crackling sound when 
adding
water  to  dry  APS.  Material that has broken down due to high humidity 
or
liquid  contamination will no longer crackle when moistened.  Persulfate 
is
consumed  in  the  polymerization  since it provides the free radicals 
that
drive  polymerization,  but  excess APS can cause oxidation of proteins 
and
nucleic  acids.   Prerunning  gels  keeps excess APS traveling ahead of 
the
sample.  BioRad recommends 50 ?l of fresh 10% APS per 10 ml of gel 
solution
for  a  final concentration of 0.05%.  I use 100 ?l of fresh 10% APS per 
10
ml of gel solution for a final concentration of 0.1%. )

(subscript: TEMED)(subscript:  (tetramethylethylenediamine))
(subscript:   TEMED  is  neurotoxic  and  extremely  flammable.   TEMED  
is
extremely destructive to mucous membranes and upper respiratory tract, 
eyes
and  skin.   Inhalation  may  be fatal.  Prolonged contact can cause 
severe
irritation or burns.)
(subscript: Use of a fume hood is recommended.)
(subscript:  TEMED  (tetramethylethylenediamine)  is  subject  to 
oxidation
which  causes  a gradual loss in catalytic activity.  It is hygroscopic 
and
accumulates  water which accelerates oxidative decomposition.  It should 
be
discarded  as  chemical  waste  when  oxidation products begin to give it 
a
yellow  color.   For this reason, it is recommended that clear tips be 
used
to add TEMED.)
(subscript:  TEMED  also effects the pH gradient of IEF gels. It 
introduces
fixed  charges  into  the gel matrix which cause gradient drift towards 
the
anode.   Non-reacted  TEMED  will  focus  at the cathode and produce a 
more
alkaline  pH  gradient.   Because  TEMED  is  only  active  as a free 
base,
polymerization   is  inhibited  at  low  pH.   BioRad  recommends  a  
final
concentration  of  1  to 10 mM TEMED.  22 ?l TEMED/100 ml of gel results 
in
1.9 mM TEMED, while 10 ?l TEMED/10 ml of gel results in 8.62 mM TEMED.  
Too
much TEMED will increase the tendency of your gel to crack during drying 
or
crumble during passive elution due to increased brittleness.)



Initiator concentration

(subscript:  These  are  initiator concentrations that I developed over 
the
course  of  many  years  of  running  sequencing  resolution RNA gels.  
The
decreasing  amounts of TEMED result in a consistent polymerization time 
and
increased elasticity.  The positions of dye markers such as bromphenol 
blue
and xylene cyanol remain consistent with published data.)
(subscript: Initiator concentration)(subscript:  for degassed gel 
solutions
)(subscript: (at Boulder's altitude, degassing is not necessary))
|----------------------------+-------+-----+-----+-----+-----+-----+----->
|                            |       |     |     |     |     |     |     |
| (subscript:  %  acryl. for | (subs | (su | (su | (su | (su | (su | (su |
| 100 ml gel)                | cript | bsc | bsc | bsc | bsc | bsc | bsc |
|                            | : 4%) | rip | rip | rip | rip | rip | rip |
|                            |       | t:  | t:  | t:  | t:  | t:  | t:  |
|                            |       | 6%) | 8%) | 10% | 12% | 14% | 15% |
|                            |       |     |     | )   | )   | )   | )   |
|                            |       |     |     |     |     |     |     |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
  >-----+-----+------|
  |     |     |      |
  | (su | (su | (sub |
  | bsc | bsc | scri |
  | rip | rip | pt:  |
  | t:  | t:  | 20%) |
  | 16% | 18% |      |
  | )   | )   |      |
  |     |     |      |
  >-----+-----+------|
|----------------------------+-------+-----+-----+-----+-----+-----+----->
|                            |       |     |     |     |     |     |     |
| (subscript:  ?l  TEMED for | (subs | (su | (su | (su | (su | (su | (su |
| native gel)                | cript | bsc | bsc | bsc | bsc | bsc | bsc |
|                            | :  24 | rip | rip | rip | rip | rip | rip |
|                            | )     | t:  | t:  | t:  | t:  | t:  | t:  |
|                            |       | 22  | 20) | 18) | 16) | 14) | 13) |
|                            |       | )   |     |     |     |     |     |
|                            |       |     |     |     |     |     |     |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
  >-----+-----+------|
  |     |     |      |
  | (su | (su | (sub |
  | bsc | bsc | scri |
  | rip | rip | pt:  |
  | t:  | t:  | 8)   |
  | 12) | 10) |      |
  |     |     |      |
  >-----+-----+------|
|----------------------------+-------+-----+-----+-----+-----+-----+----->
|                            |       |     |     |     |     |     |     |
| (subscript:  ?l  TEMED for | (subs | (su | (su | (su | (su | (su | (su |
| denat. gel)                | cript | bsc | bsc | bsc | bsc | bsc | bsc |
|                            | :  22 | rip | rip | rip | rip | rip | rip |
|                            | )     | t:  | t:  | t:  | t:  | t:  | t:  |
|                            |       | 20  | 18) | 16) | 14) | 12) | 11) |
|                            |       | )   |     |     |     |     |     |
|                            |       |     |     |     |     |     |     |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
  >-----+-----+------|
  |     |     |      |
  | (su | (su | (sub |
  | bsc | bsc | scri |
  | rip | rip | pt:  |
  | t:  | t:  | 6)   |
  | 10) | 8)  |      |
  |     |     |      |
  >-----+-----+------|


(subscript:   Initiator  concentration)(subscript:   for  non-degassed  
gel
solutions)
|----------------------------+-------+-----+-----+-----+-----+-----+----->
|                            |       |     |     |     |     |     |     |
| (subscript:  %  acryl. for | (subs | (su | (su | (su | (su | (su | (su |
| 100 ml gel)                | cript | bsc | bsc | bsc | bsc | bsc | bsc |
|                            | : 4%) | rip | rip | rip | rip | rip | rip |
|                            |       | t:  | t:  | t:  | t:  | t:  | t:  |
|                            |       | 6%) | 8%) | 10% | 12% | 14% | 15% |
|                            |       |     |     | )   | )   | )   | )   |
|                            |       |     |     |     |     |     |     |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
  >-----+-----+------|
  |     |     |      |
  | (su | (su | (sub |
  | bsc | bsc | scri |
  | rip | rip | pt:  |
  | t:  | t:  | 20%) |
  | 16% | 18% |      |
  | )   | )   |      |
  |     |     |      |
  >-----+-----+------|
|----------------------------+-------+-----+-----+-----+-----+-----+----->
|                            |       |     |     |     |     |     |     |
| (subscript:  ?l  TEMED for | (subs | (su | (su | (su | (su | (su | (su |
| native gel)                | cript | bsc | bsc | bsc | bsc | bsc | bsc |
|                            | :  26 | rip | rip | rip | rip | rip | rip |
|                            | )     | t:  | t:  | t:  | t:  | t:  | t:  |
|                            |       | 24  | 22) | 20) | 18) | 16) | 15) |
|                            |       | )   |     |     |     |     |     |
|                            |       |     |     |     |     |     |     |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
  >-----+-----+------|
  |     |     |      |
  | (su | (su | (sub |
  | bsc | bsc | scri |
  | rip | rip | pt:  |
  | t:  | t:  | 10)  |
  | 14) | 12) |      |
  |     |     |      |
  >-----+-----+------|
|----------------------------+-------+-----+-----+-----+-----+-----+----->
|                            |       |     |     |     |     |     |     |
| (subscript:  ?l  TEMED for | (subs | (su | (su | (su | (su | (su | (su |
| denat. gel)                | cript | bsc | bsc | bsc | bsc | bsc | bsc |
|                            | :  24 | rip | rip | rip | rip | rip | rip |
|                            | )     | t:  | t:  | t:  | t:  | t:  | t:  |
|                            |       | 22  | 20  | 18) | 16) | 14) | 13) |
|                            |       | )   | )   |     |     |     |     |
|                            |       |     |     |     |     |     |     |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
  >-----+-----+------|
  |     |     |      |
  | (su | (su | (sub |
  | bsc | bsc | scri |
  | rip | rip | pt:  |
  | t:  | t:  | 8)   |
  | 12) | 10) |      |
  |     |     |      |
  >-----+-----+------|


(subscript:   Initiator  concentration)(subscript:   for  non-degassed  
gel
solutions)
|----------------------------+-------+------+------+------+------+------>
|                            |       |      |      |      |      |      |
| (subscript:  %  acryl. for | (subs | (sub | (sub | (sub | (sub | (sub |
| 80 ml gel)                 | cript | scri | scri | scri | scri | scri |
|                            | : 4%) | pt:  | pt:  | pt:  | pt:  | pt:  |
|                            |       | 6%)  | 8%)  | 10%) | 12%) | 14%) |
|                            |       |      |      |      |      |      |
|----------------------------+-------+------+------+------+------+------>
  >------+------+-----+------|
  |      |      |     |      |
  | (sub | (sub | (su | (sub |
  | scri | scri | bsc | scri |
  | pt:  | pt:  | rip | pt:  |
  | 15%) | 16%) | t:  | 20%) |
  |      |      | 18% |      |
  |      |      | )   |      |
  |      |      |     |      |
  >------+------+-----+------|
|----------------------------+-------+------+------+------+------+------>
|                            |       |      |      |      |      |      |
| (subscript:  ?l  TEMED for | (subs | (sub | (sub | (sub | (sub | (sub |
| native gel)                | cript | scri | scri | scri | scri | scri |
|                            | :     | pt:  | pt:  | pt:  | pt:  | pt:  |
|                            | 20.8) | 19.2 | 17.6 | 16)  | 14.4 | 12.8 |
|                            |       | )    | )    |      | )    | )    |
|                            |       |      |      |      |      |      |
|----------------------------+-------+------+------+------+------+------>
  >------+------+-----+------|
  |      |      |     |      |
  | (sub | (sub | (su | (sub |
  | scri | scri | bsc | scri |
  | pt:  | pt:  | rip | pt:  |
  | 12)  | 11.2 | t:  | 8)   |
  |      | )    | 9.6 |      |
  |      |      | )   |      |
  |      |      |     |      |
  >------+------+-----+------|
|----------------------------+-------+------+------+------+------+------>
|                            |       |      |      |      |      |      |
| (subscript:  ?l  TEMED for | (subs | (sub | (sub | (sub | (sub | (sub |
| denat. gel)                | cript | scri | scri | scri | scri | scri |
|                            | :     | pt:  | pt:  | pt:  | pt:  | pt:  |
|                            | 19.2) | 17.6 | 16)  | 14.4 | 12.8 | 11.2 |
|                            |       | )    |      | )    | )    | )    |
|                            |       |      |      |      |      |      |
|----------------------------+-------+------+------+------+------+------>
  >------+------+-----+------|
  |      |      |     |      |
  | (sub | (sub | (su | (sub |
  | scri | scri | bsc | scri |
  | pt:  | pt:  | rip | pt:  |
  | 10.4 | 9.6) | t:  | 6.4) |
  | )    |      | 8)  |      |
  |      |      |     |      |
  >------+------+-----+------|


(subscript:   Initiator  concentration)(subscript:   for  non-degassed  
gel
solutions )
|----------------------------+-----+------+------+------+------+------>
|                            |     |      |      |      |      |      |
| (subscript:  %  acryl. for | (su | (sub | (sub | (sub | (sub | (sub |
| 30 ml gel)                 | bsc | scri | scri | scri | scri | scri |
|                            | rip | pt:  | pt:  | pt:  | pt:  | pt:  |
|                            | t:  | 6%)  | 8%)  | 10%) | 12%) | 14%) |
|                            | 4%) |      |      |      |      |      |
|                            |     |      |      |      |      |      |
|----------------------------+-----+------+------+------+------+------>
  >------+------+------+------|
  |      |      |      |      |
  | (sub | (sub | (sub | (sub |
  | scri | scri | scri | scri |
  | pt:  | pt:  | pt:  | pt:  |
  | 15%) | 16%) | 18%) | 20%) |
  |      |      |      |      |
  >------+------+------+------|
|----------------------------+-----+------+------+------+------+------>
|                            |     |      |      |      |      |      |
| (subscript:  ?l  TEMED for | (su | (sub | (sub | (sub | (sub | (sub |
| native gel)                | bsc | scri | scri | scri | scri | scri |
|                            | rip | pt:  | pt:  | pt:  | pt:  | pt:  |
|                            | t:  | 7.2  | 6.6) | 6.0) | 5.4) | 4.8) |
|                            | 7.8 | )    |      |      |      |      |
|                            | )   |      |      |      |      |      |
|                            |     |      |      |      |      |      |
|----------------------------+-----+------+------+------+------+------>
  >------+------+------+------|
  |      |      |      |      |
  | (sub | (sub | (sub | (sub |
  | scri | scri | scri | scri |
  | pt:  | pt:  | pt:  | pt:  |
  | 4.5) | 4.2) | 3.6) | 3.0) |
  |      |      |      |      |
  >------+------+------+------|
|----------------------------+-----+------+------+------+------+------>
|                            |     |      |      |      |      |      |
| (subscript:  ?l  TEMED for | (su | (sub | (sub | (sub | (sub | (sub |
| denat. gel)                | bsc | scri | scri | scri | scri | scri |
|                            | rip | pt:  | pt:  | pt:  | pt:  | pt:  |
|                            | t:  | 6.6) | 6.0) | 5.4) | 4.8) | 4.2) |
|                            | 7.2 |      |      |      |      |      |
|                            | )   |      |      |      |      |      |
|                            |     |      |      |      |      |      |
|----------------------------+-----+------+------+------+------+------>
  >------+------+------+------|
  |      |      |      |      |
  | (sub | (sub | (sub | (sub |
  | scri | scri | scri | scri |
  | pt:  | pt:  | pt:  | pt:  |
  | 3.9) | 3.6) | 3.0) | 2.4) |
  |      |      |      |      |
  >------+------+------+------|


(subscript:  Use  of  more  than recommended volume of TEMED will result 
in
brittle  gels.   Note  that the volume of TEMED decreases 2 ?l for every 
2%
increase in gel concentration in a 100 ml gel stock.  )

(subscript:  The  properties of the gel depend on the concentrations of 
APS
and  TEMED.   Increasing  the  amount  of APS/TEMED leads to an increase 
in
chain  initiation  events  and  a  corresponding  decrease in chain 
length,
resulting  in gel turbidity and decreased gel elasticity.  Excess 
APS/TEMED
can  also  oxidize  sulfhydryl-containing  proteins  and  result in 
altered
buffer  pH.   Excess  TEMED can increase buffer pH, react with proteins 
and
alter banding patterns.  Excess APS acts as a buffer between pH 8 and 9.  
)
(subscript:  Reducing  the  amount  of  APS/TEMED  results  in longer 
chain
length,  lower  turbidity  and  greater  elasticity.   These  are 
desirable
properties.   Low  percentage  acrylamide gels are most sensitive to 
excess
initiator  concentrations,  producing  soft mushy gels if too much TEMED 
is
used.  )
(subscript: If concentrations of APS/TEMED are too low, polymerization 
will
be  too slow and allow air to enter the monomer and result in gels that 
are
too porous and mechanically weak.  The unpolymerized monomer can react 
with
)(subscript:  a)(subscript:  amino, sulfhydryl and phenolic hydroxyl 
groups
of proteins.)

(subscript: Tim Fitzwater)





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